A new target for diagnosis and treatment of infected and non-infected malignant tumors and its intervention application

Abstract

The present application belongs to the field of biomedical technology, and relates to development and application of tumor markers, in particular to a detection method and evaluation standard of an acid response sequence of YY1 as a tumor marker, and potential intervention application of the acid response sequence of YY1 protein and YY1 protein in development of anti-tumor targeted drugs. The present application discloses a polypeptide comprising an MH region of YY1 amino acids 70-80. The present application also relates to application of the polypeptide, a corresponding polypeptide recognition reagent and a kit. The acid response sequence of YY1 can be used as a tumor grading and prognosis detection molecular marker, and a targeted inhibitor thereof is used for potential application in anti-tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the development and application of tumor markers. Specifically, it relates to the detection method and evaluation standard of the acid response sequence of YY1 as a tumor marker, as well as the presence of the acid response sequence of YY1 protein and its potential intervention application in the development of anti-tumor targeted drugs. Background Technology

[0002] YY1 (YinYang1, NF-E1, UCRBP, or CF1) was discovered by Professor Yang Shi in 1991 [1,2]. It is a highly conserved zinc finger transcription factor [3-5], belonging to the human GLI-Kruppel protein family [2], and is involved in transcriptional activation, transcriptional repression, transcriptional initiation, and chromatin modification. YY1 is widely expressed in mammalian cells and tissues, and its binding sites are found in about 10% of human genes and repetitive sequences [6]. The open reading frame of human YY1 cDNA encodes a 414-amino acid protein with a molecular weight of 44 kDa. However, possibly due to the structure of the YY1 protein, its molecular weight on SDS gel is 65-68 kDa. The C-terminus of YY1 is a highly conserved zinc finger domain that binds to sequence-specific DNA to exert activation or repression functions. The amino acid 43-53 region near the N-terminus consists of 11 consecutive acidic amino acids, and the amino acid 70-80 region consists of 11 consecutive histidines, both located in the activation domain of YY1; the middle amino acid 154-201 region of YY1 has a high proportion of glycine and alanine residues, located in the inhibition domain of YY1. Therefore, there are two inhibition domains in YY1, one with the zinc finger domain and the other is the glycine-rich amino acid 154-226 region [7]. YY1 participates in the regulation of various biological processes such as cell differentiation, embryonic development and tumorigenesis [8-13]. It can regulate the life cycle of various oncogenic viruses and regulate tumor development and progression. YY1 inhibits the transcription of HPV genome E6 and E7 genes and inhibits the activation of EBV and KSHV cleavage replication switch genes. Therefore, YY1 may play a role in inhibiting tumor progression in tumors of oncogenic human papillomavirus (HPV) [14-16], Epstein-Barr virus (EBV) [17-20], and Kaposi's sarcoma virus (KSHV) [21-23]. Numerous studies have shown that YY1 can promote the development of HBV-related liver cancer [24-27], hepatocellular carcinoma [28-32], colorectal cancer [33-40], glioma [41-43], lung cancer [44-46], neuroblastoma

[47] , and Wilms' tumor

[48] through multiple mechanisms, and is positively correlated with poor patient prognosis; it can also inhibit the development of esophageal squamous cell carcinoma

[49] ; however, its pro- or anti-cancer effects in nasopharyngeal carcinoma [50,51] and breast cancer [52-54] are controversial and require further investigation. Many studies have also shown that YY1 expression levels are closely related to tumor grade and prognosis, making it a potential prognostic indicator and therapeutic target for tumors.

[0003] The tumor microenvironment (TME) is a complex dynamic system composed of tumor cells and their surrounding fibroblasts, immune cells, pericytes, adipocytes, endothelial cells, neurons, and extracellular matrix

[55] . The extracellular environment of normal cells is a neutral environment with a pH of 7.4, while tumor cells, especially solid tumors, produce acid due to the "Warburg effect," particularly lactic acid and carbon dioxide, leading to acidification of the tumor microenvironment. At physical pH, more than 99% of lactic acid dissociates into lactate anions and hydrogen ions (H+). Lactate dehydrogenase is a class of NAD+-dependent enzymes. The activated state of LDH is a homotetrameric molecule composed of two different subunits: lactate dehydrogenase A (LDHA) and lactate dehydrogenase B (LDHB). LDHA and LDHB play a role in regulating the bidirectional conversion of pyruvate and lactate in tumor cell metabolism. LDHA has a high affinity for pyruvate and preferentially converts pyruvate to lactate and NADH to NAD(+) under anaerobic conditions. LDHB has a high affinity for lactate and preferentially converts lactate to pyruvate and NAD(+) to NADH when oxygen is sufficient

[56] .

[0004] Lactate is recruited to tumor sites by a large number of immune cells and plays a key role in disrupting immune surveillance. Lactate is considered a key component that leads to the creation of an immunosuppressive tumor microenvironment

[57] . Metabolic competition between cancer cells and immune cells (mainly T and natural killer cells, NK cells) is an important factor in tumor progression. The Warburg metabolic effect competitively provides an advantage to cancer cells, leading to the consumption of extracellular glucose and the release of large amounts of lactate, thereby causing dysfunction of tumor-infiltrating T cells

[58] . Pancreatic cancer cells that highly express MCT4 also have an adverse effect on the surrounding immune environment

[59] . In melanomas that highly express LDHA, lactate accumulation inhibits the survival of T cells and NK cells, leading to tumor immune escape

[60] . Dendritic cells and DC cells in the tumor microenvironment are also affected by lactate accumulation. The accumulation of endogenous lactate causes DC cells to differentiate into a tolerant phenotype, leading to immune tolerance. Excessive acidification interferes with the production of inflammatory cytokines, which are essential for Th1 cell polarization and differentiation of inflammatory DC cells

[61] . Lactic acid promotes antigen degradation and inhibits the ability of MHC class I antigens to present, thereby preventing DC cells from initiating antitumor responses

[62] .

[0005] Lactic acid also plays a signaling role in driving cancer immune evasion. Lactic acid acts as a pro-inflammatory mediator by promoting the transcription of IL-23 in monocytes / macrophages mediated by Toll-like receptors (TLRs), maintaining the secretion of IL-17 dependent on IL-23, and polarizing the immune response toward the Th17 type

[63] . Lactic acid plays a key role in signal transduction by inducing the expression of vascular endothelial growth factor and inducing the polarization of M2 type TAMs

[64] .

[0006] Lactate can serve as an extracellular ligand, among which G protein-coupled receptor 81 (GPR81, also known as HCAR1) is a lactate receptor

[65] . Activated GPR81 can maintain tumor growth and metastasis by modulating lactate response mechanisms

[66] , and the lactate / GPR81 pathway also plays a role in suppressing immune surveillance

[67] . In addition, GPR132 is a lactate sensor / receptor specifically expressed in macrophages, which promotes tumor metastasis by regulating the interaction between breast tumors and macrophages through promoting M2 phenotypic polarization[68,69]. Lactate binds directly to oxygen sensors (such as NDRG3), preventing their binding to and degradation of PHD2, increasing the stability of NDRG3 protein, and promoting angiogenesis and cell growth

[70] .

[0007] Lactic acid plays a crucial role in various biological processes, including the regulation of angiogenesis, hypoxia, macrophage polarization, T cell activation, and tumor immune surveillance, and is closely associated with a variety of diseases, including tumor proliferation, sepsis, and autoimmune diseases [60,64,71]. The lactic acid microenvironment promotes the occurrence, development, invasion, and metastasis of various tumors through metabolic regulation, immune regulation, and many other aspects. Lactic acid is not only a metabolite but also an important participant in cellular metabolism. As a signaling molecule, lactic acid can directly bind to the intracellular receptor MAVS, thereby inhibiting the production of RLR-mediated interferon, and plays a key role in improving host immunity, viral clearance, and cancer immune surveillance. At the same time, lactic acid acts as a bridge connecting energy metabolism and innate immunity

[72] . Lactic acid causes histone lactation modification, regulates the expression of related genes, and polarizes M2 macrophages [71,73]. Smad5 maintains cellular bioenergy homeostasis by responding to changes in intracellular pH through nucleocytoplasmic translocation

[74] . So, can YY1, an important protein widely expressed in mammalian cells and tissues, respond to the acidic microenvironment of lactate and thus affect the malignant progression of various tumors?

[0008] The massive production of acidic metabolites and the enhanced export of acid to the extracellular environment lead to the continuous acidification of the tumor microenvironment. Elevated lactate concentration is a good indicator of tumor metabolic adaptation, and elevated expression of lactate dehydrogenase A (LDHA) is associated with poor prognosis in various tumors

[60] . Since lactate can promote tumor development, many cancer treatment strategies have been developed targeting the molecular mechanisms of lactate production and transport. Targeting lactate metabolism and transport is a promising approach for cancer treatment. Increased expression of MCT1 and MCT4 is associated with poor prognosis in prostate cancer, colorectal cancer, neuroblastoma, and gastrointestinal stromal tumors. MCT1 inhibitors can effectively inhibit tumor growth. The inhibitor AZ3965, which targets MCT1 / 2, is effective in treating Burkitt lymphoma, breast cancer, gastric cancer, and small cell lung cancer (SCLC) models expressing MCT1 [75,76]. Studies have reported that MCT4 may be a potential target for the treatment of colorectal cancer, glioblastoma, breast cancer, and prostate cancer [77-80]. The inhibitor AZ93, which targets MCT4, is highly effective and selective and is currently being used in preclinical studies

[81] . Compounds 7-amino-carboxylic acid coumarins (7ACCs) inhibit extracellular lactate uptake by interfering with MCT1 / 4, thus hindering the transport of mitochondrial pyruvate and promoting the accumulation of intracellular pyruvate

[82] . Therapeutic treatments include gossypol (AT-101) derivatives FX-11, free flavins, and n-hydroxyindole compounds that target the bidirectional conversion of pyruvate and lactate mediated by LDH enzymes

[83] . However, since metabolic reprogramming plays a role in tumorigenesis and therapeutic adaptation, the transformation of LDH expression subtypes may occur during tumor progression

[84] , which may affect the therapeutic effect.

[0009] Currently, a variety of malignant tumor diagnosis and treatment strategies based on the acidic tumor microenvironment have been developed, such as pH-responsive iron oxide nanoparticles

[85] , NIR-II nanopermeation system (FEAD1) that depends on tumor microenvironment activation

[86] , pH-responsive AuNCs@MOF-DOX nanoprobes

[87] , and polyethylene glycol-modified hyaluronic acid nanodrug delivery system

[88] and other nanoparticle technologies. Summary of the Invention

[0010] The purpose of this invention is to expand the application scope of YY1 as a biomarker for tumor grading and prognostic evaluation.

[0011] A further objective of this invention is to provide the presence of an acid-responsive sequence in the YY1 protein and its application in targeted drug development.

[0012] This invention is based on extensive previous research findings that exogenous lactic acid and acidic conditions regulate YY1 protein expression levels. Further experiments revealed that endogenous lactic acid produced by cell metabolism downregulates YY1 protein expression levels, thereby promoting the rapid growth and tumorigenicity of EBV-related tumor cells. YY1 protein expression levels were significantly reduced in PBMCs of lymphoma patients with high LDH and in tissue samples from high-grade cervical cancer patients, suggesting a correlation between YY1 protein expression levels and tumor malignancy. YY1 protein expression levels were negatively correlated with the malignancy of various clinical virus-related tumor samples, further indicating that the YY1 protein acid-responsive sequence can serve as a biomarker for potential tumor grading and prognostic evaluation. The acidic lactic acid microenvironment promotes the nuclear degradation of YY1. Site-directed mutagenesis identified the YY1 protein acid-responsive sequence as the MH region located at amino acid positions 70-80. Mutations in the YY1 protein acid-responsive sequence MH significantly reduce and enhance the stability of YY1 under acidic conditions and significantly inhibit the in vitro clonogenic ability of HPV-infected cells. Stable expression of the MH site mutation can significantly inhibit the formation of HeLa clones in HPV-transformed cell lines. This invention is based on the above research.

[0013] On one hand, the present invention provides a polypeptide comprising amino acids YY1 at positions 70-80, hereinafter referred to as YY1-MH. Preferably, its sequence is shown in SEQ ID NO 4.

[0014] In this invention, YY1 refers to YinYang1, NF-E1, UCRBP, or CF1 [1,2]. Preferably, the polypeptide is YY1, i.e., the full length of YY1. Preferably, its sequence at positions 70-80 is as shown in mH.

[0015] The experimental results of this invention demonstrate the correlation between YY1's response to the acidic lactate microenvironment and the malignancy of oncovirus-associated tumors. Specifically, the acidic lactate microenvironment promotes the translocation of YY1 from the nucleus to the cytoplasm and reduces the expression level of YY1 protein. Furthermore, the phenomenon of YY1 responding to the acidic lactate microenvironment is prevalent in various oncovirus-associated tumors, and the response of YY1 to the acidic lactate microenvironment is closely related to the malignancy grade of clinical tumors.

[0016] On the other hand, the present invention provides the application of the aforementioned polypeptide YY1-MH in the preparation of drugs for diagnosing or treating tumors.

[0017] The drugs mentioned include agents for predicting tumor grade or prognosis, diagnosing and predicting tumor grade or prognosis, and treating tumors.

[0018] The YY1 protein acid response sequence MH described in this invention can be used as a candidate drug intervention target for YY1 protein, for screening small molecule chemical drugs or small molecule peptide drugs, and further for the development of anti-tumor drugs.

[0019] For example, the aforementioned polypeptide YY1-MH can be used as a biomarker for predicting tumor grade or prognosis.

[0020] The application includes the following steps: detecting the localization of the above-mentioned polypeptide YY1-MH; and / or its stability;

[0021] The localization or stability of the aforementioned polypeptide YY1-MH can be used as an indicator for the diagnosis or treatment of tumors.

[0022] Studies have shown that in tumor samples, the polypeptide YY1-MH exhibits nucleus extrusion, and YY1 undergoes degradation. Therefore, the content of YY1-MH in the cytoplasm is higher than that in the cytoplasm of normal tissues, and can be used as an indicator for diagnosing tumors.

[0023] In this invention, the polypeptide YY1-MH can be used to select drugs for the diagnosis or treatment of tumors.

[0024] Preferably, the tumor is caused by EBV or HPV, or is one or more of lymphoma, cervical cancer, liver cancer, or colorectal cancer.

[0025] EBV and HPV are viruses that cause nasopharyngeal carcinoma and cervical cancer, respectively.

[0026] In another aspect, the present invention provides a peptide recognition reagent capable of recognizing peptide YY1-MH. Preferably, the peptide recognition reagent is an antibody, or the recognition site of a conjugated antibody.

[0027] This invention also provides a method and evaluation criteria for detecting YY1 acid response sequences that can be used to predict tumor grade and prognosis, comprising the following steps:

[0028] (1) Under acidic microenvironment conditions, YY1 undergoes nuclear translocation, resulting in a decrease in total protein expression levels. Mutations in the acid-responsive sequence of YY1 can effectively inhibit nuclear translocation of YY1 under acidic microenvironment conditions. The acid-responsive sequence of YY1 is located at amino acid positions 70-80 of YY1.

[0029] (2) Using YY1-specific recognition antibodies against the above-mentioned YY1 different mutation site sequences, the stability of the above-mentioned YY1 different mutation site sequences was detected and applied by Western blot at different tumor tissue and cell levels;

[0030] (3) The acid response sequence mutation of YY1 can effectively inhibit the in vitro proliferation of EBV and HPV-related tumor cells.

[0031] Furthermore, the present invention also provides a kit for diagnosing or treating tumors, wherein the kit contains one or more of the following substances: a recognition reagent capable of recognizing polypeptide YY1-MH, polypeptide YY1-MH, a reagent capable of mutating position 70-80 of polypeptide YY1, a substance that inhibits glucose, and a substance that inhibits lactic acid.

[0032] This invention belongs to the biomedical field and relates to the acid-responsive sequence of YY1, a novel biomarker for predicting tumor grade and prognosis. Specifically, it includes a detection method and evaluation criteria for the YY1 protein acid-responsive sequence as a tumor biomarker, the presence of an acid-responsive sequence in the YY1 protein, and its application in the development of anti-tumor targeted drugs. The acid-responsive sequence of YY1 described in this invention is the first internationally revealed sequence showing that YY1 responds to an acidic tumor microenvironment. This phenomenon of YY1 responding to acid exists in tumor tissues and cells from various sources, including cervical cancer cells and tissues, lymphoma cells and PBMCs from lymphoma patients, liver cancer cells, and colorectal cancer cells. Under acidic microenvironment conditions, YY1 protein shifts from the nucleus to the cytoplasm, and the total expression level of YY1 protein decreases, promoting the in vitro and in vivo tumorigenicity of EBV and HPV-related tumor cells; mutations in the YY1 acid-responsive sequence can effectively inhibit the proliferation of EBV and HPV-related tumor cells. The expression level of YY1 protein is negatively correlated with the malignancy of lymphoma, cervical cancer, and liver cancer in clinical practice. The YY1 acid response sequence provided by this invention can be used as a molecular marker for tumor grading and prognosis detection, and its targeted inhibitors have potential applications in anti-tumor therapy.

[0033] For ease of understanding, the present invention will be described in detail below with reference to specific drawings and embodiments. It should be particularly noted that the specific examples and drawings are for illustrative purposes only. Obviously, those skilled in the art can make various modifications and changes to the present invention within the scope of this invention based on the description herein, and these modifications and changes are also included within the scope of this invention. Attached Figure Description

[0034] Figure 1 As shown, lactic acid (LA) reduced the expression level of YY1 protein in EBV-infected cells, KSHV-infected cells, EBV and KSHV double-negative cells, cervical cancer cell lines, colorectal cancer cell lines, liver cancer cell lines, PBMCs from lymphoma patients, and cervical cancer tissues. F represents the YY1 protein expression level in PBMCs from lymphoma patients, and G represents the YY1 protein expression level in tissues from cervical cancer patients.

[0035] Figure 2As shown: Intracellular lactate promotes the downregulation of YY1 expression.

[0036] Figure 3 As shown, the lactic acidic microenvironment promotes the malignant proliferation of EBV-related lymphomas by downregulating the expression of YY1.

[0037] Figure 4 As shown, the lactic acid-acidic microenvironment promotes the malignant proliferation of HPV-related cervical cancer by downregulating the expression of YY1.

[0038] Figure 5 As shown, YY1 exhibits a response to lactate nucleotransfer.

[0039] Figure 6 As shown: The MH mutation in the YY1 acid response sequence hinders the nuclear degradation of YY1.

[0040] The LAR1 sequence is: WT,EEEEEDDDDED (SEQ ID NO 1),mE,AAAAAAAAAAA (SEQ ID NO 2).

[0041] LAR2 sequence: WT, HHHHHHHHHHH (SEQ ID NO 3), mH, AAAAAAAAAAA (SEQ ID NO 4).

[0042] NES1 sequence: WT, IVELHEIEV (SEQ ID NO 5), mNES1, AVEAHEAEA (SEQ ID NO 6).

[0043] NES2 sequence: WT, IKTLEGEFSV (SEQ ID NO 7), mNES2, AKTAEGEASA (SEQ ID NO 8).

[0044] NES3 sequence: WT, LDFNLRTHVRI (SEQ ID NO 9), mNES3, ADANLRTHARA (SEQ ID NO 10).

[0045] Figure 7 As shown: The MH mutation in the YY1 acid-responsive sequence inhibits the in vitro clonogenic ability of HPV-positive cells. Detailed Implementation

[0046] Example 1: Regulation of YY1 protein expression level under lactic acid conditions.

[0047] 1. Exogenous lactic acid acidic conditions regulate the expression level of YY1 protein in EBV, KSHV positive or negative tumor cell lines of different cell origins.

[0048] EBV-exposed cell lines LCL and B95.8, as well as the EBV / KSHV double-negative cell line DG75, were treated for 36 h with 10 mM LA (containing both lactate anions and hydrogen ions), lactate (NaLA, containing lactate anions), and H+ (containing only hydrogen ions), respectively. Cells were collected, proteins were extracted, and Western blotting was used to detect the protein expression levels of YY1 and YY2. The results are as follows: Figure 1 As shown in Figure A, LA treatment significantly reduced the expression levels of YY1 protein in LCL and DG75 cells, and also reduced the expression levels of YY1 protein in B95.8 cells. Simultaneously, both NaLA and H+ treatments reduced the expression levels of YY1 protein in LCL, B95.8, and DG75 cells. In LCL and B95.8 cells, LA treatment significantly reduced the expression level of YY2 protein, and both NaLA and H+ treatments reduced the expression level of YY2 protein. In DG75 cells, H+ treatment significantly reduced the expression level of YY2 protein, while LA and NaLA treatments had no effect on the expression level of YY2 protein.

[0049] Both EBV and KSHV belong to the Gamma Herpesvirus subfamily and are human oncogenic viruses with similar life cycles: a latent infection period and a lytic replication period. To verify whether LA, NaLA, or H+ treatment would reduce the expression levels of YY1 and YY2 proteins in the KSHV-treated cell line BCBL1, the KSHV-treated cell line BCBL1, the EBV-treated cell line Akata-EBV, and the KSHV / EBV double-negative cell line Akata were selected and treated with LA, NaLA, or H+ for 12 h or 24 h, respectively. Western blotting was used to detect the protein expression levels of YY1 and YY2. Figure 1 As shown in Figure B, in the KSHV-treated cell line BCBL1, the EBV-treated cell line Akata-EBV, and the KSHV / EBV double-negative cell line Akata, treatment with LA, NaLA, or H+ for 12 h or 24 h reduced the protein expression levels of YY1 and YY2 to varying degrees, with LA treatment showing a more significant effect than NaLA or H+ treatment. To further verify whether LA treatment affected the expression levels of YY1 and YY2 in the KSHV-treated endothelial cell line iSLK-Bac16 and the KSHV-negative endothelial cell line iSLK, iSLK and iSLK-Bac16 were treated with 10 mM and 20 mM LA for 24 h. Figure 1 As shown in Figure C, 20 mL MLA significantly reduced the protein expression level of YY1, while having little effect on the protein expression level of YY2. In summary, lactic acid (LA) reduced the protein expression level of YY1 in EBV-treated cells, KSHV-treated cells, and EBV and KSHV double-negative cells.

[0050] 2. Exogenous lactic acid acidification regulates the expression level of YY1 protein in HPV-positive or HPV-negative tumor cell lines of different cell origins.

[0051] Further, three cervical cancer cell lines, C33A, HeLa, and Caski (HeLa and Caski were HPV-infected cell lines), three colorectal cancer cell lines, HCT116, SW480, and RKO, and three liver cancer cell lines, 7721, HLE, and HLF, were selected. All three were treated with different concentrations of LA (0 mM, 10 mM, 20 mM, and 30 mM) for 24 h. Western blotting was used to detect the expression level of YY1 protein. Figure 1 As shown in D, LA promotes the downregulation of YY1 protein expression. The degree of YY1 response to LA varies in different cell lines, but the reduction in YY1 expression becomes more pronounced with increasing LA concentration.

[0052] 3. Predicting the malignancy of clinical B-cell lymphoma samples using YY1 protein expression levels:

[0053] Elevated expression of lactate dehydrogenase A (LDHA) is associated with poor prognosis in patients with various tumors

[60] . Clinically, the LDH level in the serum of patients is detected using an LDH detection kit, with LDH < 250 U / L (4.12 μkat / L) defined as the normal reference. To detect whether lactate promotes low expression of YY1 in lymphoma patients, we collected samples from 11 lymphoma patients with LDH < 250 U / L (4.12 μkat / L) and 13 lymphoma patients with LDH > 500 U / L (4.12 μkat / L), extracted RNA from PBMCs for reverse transcription and qPCR, and detected the mRNA levels of YY1 and YY2, such as... Figure 1 As shown in Figure E, the YY1 mRNA expression level in samples from lymphoma patients with LDH > 500 U / L (4.12 μkat / L) was significantly lower than that in samples from lymphoma patients with LDH < 250 U / L (4.12 μkat / L). This indicates that lactate downregulates the mRNA expression levels of YY1 and YY2 in lymphoma patients. To further determine the correlation between YY1 protein expression levels and lymphoma malignancy, we collected clinical blood samples from 4 lymphoma patients with LDH < 250 U / L (4.12 μkat / L) and 5 lymphoma patients with LDH > 250 U / L (4.12 μkat / L). Protein was extracted from PBMCs and detected using the same Western blot method for YY1 protein. The results showed... Figure 1 The protein expression level of YY1 in lymphoma PBMCs with LDH > 250 U / L (4.12 μkat / L) was significantly lower than that in lymphoma PBMCs with LDH < 250 U / L (4.12 μkat / L), indicating that lactate downregulates the protein expression level of YY1 in lymphoma patients.

[0054] 4. Predicting the malignancy of clinical cervical cancer samples using YY1 protein expression levels:

[0055] To further determine the correlation between YY1 protein expression levels and the malignancy of cervical cancer, we collected 6 clinically HPV-positive cervical squamous cell carcinoma (SCC) tissues, 7 HPV-negative cervical intraepithelial lesions or malignant tumors (NILM), and 2 HPV-positive atypical squamous cell carcinoma tissues of indeterminate significance (ASC-US) for YY1 protein expression. Western blot results showed that the YY1 protein expression level in all cervical cancer tissues was significantly lower than that in the HPV-positive atypical squamous cell carcinoma of indeterminate significance (ASC-US), with the highest YY1 protein expression level observed in the 7 HPV-negative cervical intraepithelial lesion or malignant tumor (NILM) tissues. Figure 1 G) reflects a negative correlation between the expression level of YY1 and the malignancy of cervical cancer. The higher the malignancy of malignant tumors, especially solid tumors, the higher the lactic acid production, further confirming the phenomenon that lactic acid reduces the expression level of YY1 protein in clinical patients.

[0056] The specific steps are as follows:

[0057] 1) Preparation of protein samples from cells and clinical tissues:

[0058] Cell sample lysis preparation:

[0059] a. Collect the cell suspension into a 15 mL centrifuge tube, centrifuge at 2000 rpm for 5 min, and discard the supernatant.

[0060] b. Add 3 mL of pre-cooled 1×PBS, resuspend the cells, centrifuge at 2000 rpm for 5 min, wash 3 times, and discard the supernatant.

[0061] c. Add an appropriate amount of RIPA protein lysis buffer containing four protease inhibitors to a 15 mL centrifuge tube, resuspend the cells, and lyse on ice for 30 min, vortexing once every 5 min during the process to ensure complete lysis.

[0062] d. Transfer to a pre-chilled 1.5 mL EP tube, centrifuge at 14500 rpm for 5 min at 4 °C, transfer the supernatant to a new pre-chilled 1.5 mL EP tube, and place on ice to determine the protein concentration.

[0063] Tissue sample preparation:

[0064] a. Thaw a frozen tissue sample the size of a soybean on ice, cut it into rice-grain-sized pieces with clean scissors, place it in a 2ml round-bottom EP tube, add 4 small steel balls and an appropriate amount of RIPA lysis buffer.

[0065] b. Place the tissue sample into the module of a pre-cooled tissue homogenizer and homogenize it intermittently twice at 60 Hz for 30 seconds.

[0066] c. Centrifuge the lysed cell or tissue homogenate at 14500 rpm, 4°C, for 5 min.

[0067] d. Transfer the protein supernatant to a new EP tube, add 6×SDS loading buffer containing DDT, and heat in a 100°C metal bath for denaturation for 10 min.

[0068] 2) Procedure for Western blotting protein immunoblotting:

[0069] The denatured protein samples were loaded onto 9% SDS-PAGE gels and subjected to gel electrophoresis.

[0070] Electrophoresis is performed at a constant voltage of 80V for 40 minutes, or at 100V for approximately 90 minutes, until the bromophenol blue indicator band reaches the bottom of the separating gel, at which point electrophoresis is stopped.

[0071] Immerse the NC membrane (6cm x 9cm) in 1× membrane equilibration solution for 1 minute, soak the gel in distilled water for 1 minute, remove the transfer clip, and install the transfer clip in the following order: positive electrode - dry sponge pad - NC membrane - gel - dry sponge pad - negative electrode.

[0072] Using the eBlotTML1 rapid wet transfer instrument, transfer the film for 10-20 minutes according to the set program.

[0073] Blocking: Prepare 5% skim milk (PBS preparation) 15 minutes in advance, pre-shake on a shaker for 10-15 minutes, remove the NC membrane to be blocked after protein transfer, and incubate in 5% skim milk at room temperature for 0.5-1 hour.

[0074] Wash the membrane: Wash 3 times with 1×TBST, 5 min each time.

[0075] Primary antibody incubation: Prepare mouse YY1 primary antibody using 1×TBS buffer at a dilution ratio of 1:10000. Place the NC membrane in the prepared antibody and incubate overnight on a shaker at 4°C.

[0076] Wash the membrane: Wash 3 times with 1×TBST, 5 min each time.

[0077] Secondary antibody incubation: Use secondary anti-mouse fluorescent secondary antibody 800 (diluted 1:10000 in PBS) and incubate at room temperature in the dark for 1 hour.

[0078] Wash the membrane: Wash 3 times with 1×TBST, 5 min each time.

[0079] Imaging and analysis were performed using the Odyssey dual-color infrared laser imaging system.

[0080] 3) Reagent preparation:

[0081] RIPA lysis buffer: 1% NP-40, 2mM EDTA, 150mM NaCl, 50mM Tris (pH 7.6), (1MmPMSF, 1g / ml Aprotinin, 1g / ml leupeptin and 1g / ml pepstatin).

[0082] 4) Isolation of human peripheral blood mononuclear cells (PBMCs)

[0083] Sample processing: Transfer 25 mL of whole blood obtained from the sampling into a 50 mL centrifuge tube, filter it with a 40 μm filter to remove the coagulated blood sample;

[0084] Take a 50ml centrifuge tube and add 15mL of Ficoll solution. Gently add the filtered blood to the top of the Ficoll solution, being very careful to avoid mixing the two solutions.

[0085] 400g, centrifuged for 30 minutes, with the centrifuge acceleration set to 5 and deceleration to 0.

[0086] The cell layer containing PBMCs is white. Carefully aspirate this layer of cells into another clean 15mL centrifuge tube.

[0087] Add 1×PBS, incubate at 2000 rpm for 7 min, then discard the supernatant and set aside.

[0088] 5) RNA extraction from human peripheral blood mononuclear cells (PBMCs)

[0089] Add 1 mL of Trizol to the collected PBMCs to resuspend the lysed cells, mix thoroughly, transfer to a 1.5 mL EP tube, and incubate at room temperature for 3-5 min.

[0090] Add 200 μL of chloroform, invert and mix several times, and let stand on ice for 5 min. Centrifuge at 12000 rpm for 15 min at 4℃.

[0091] Slowly aspirate approximately 400 μL of the aqueous phase containing RNA into a new pre-chilled 1.5 mL EP tube. Avoid contact with the protein layer in the middle during aspiration.

[0092] Add 500 μL of isopropanol to 400 μL of RNA aqueous solution, invert 15-20 times to mix thoroughly, and incubate on ice for 10 min. Centrifuge at 12000 rpm for 15 min at 4℃, and discard the supernatant.

[0093] Invert the centrifuge tube onto absorbent paper to further remove residual liquid; white RNA precipitate will be visible at the bottom.

[0094] Add 1 mL of 70% ethanol to wash the precipitate. Centrifuge at 12000 rpm for 10-15 min at 4℃.

[0095] Discard the supernatant, invert the centrifuge tube onto filter paper, and let it dry for 10 minutes to allow the precipitate to set.

[0096] Dissolve the RNA in 10-15 μL of RNase-free ddH2O and determine the RNA concentration using Nanodrop.

[0097] All of the above processes use RNase-free EP tubes, pipette tips, and reagents.

[0098] 6) Protein extraction from human peripheral blood mononuclear cells (PBMCs)

[0099] Add 50 μL of RIPA protein lysis buffer containing four protease inhibitors to the collected PBMCs, resuspend the cells, and lyse on ice for 30 min, vortexing once every 5 min during the lysis to ensure complete lysis.

[0100] Centrifuge at 14500 rpm for 5 min at 4℃, transfer the supernatant to a new pre-cooled 1.5 mL EP tube, place on ice, and measure the protein concentration.

[0101] Example 2: Endogenous lactate produced by cell metabolism downregulates the expression level of YY1 protein.

[0102] The above experiments demonstrated that exogenous lactate supplementation reduces the protein expression level of YY1 in tumor cells. Therefore, we wanted to investigate whether endogenous lactate produced by cell metabolism plays a role in reducing YY1 protein expression. The main source of intracellular lactate is glucose. We treated BJAB, HeLa, and LCL cells with different concentrations of glucose for 24 hours, and used an intracellular lactate detection kit to detect the intracellular lactate content. Figure 2 As shown in A, glucose promotes intracellular lactate production in a dose-dependent manner.

[0103] To demonstrate that lactate production is indeed generated from the metabolism of added glucose, we used the non-metabolizable glucose analog 2-deoxy-D-glucose (2-DG) as a control, treating BJAB, HeLa, and LCL cells for 24 hours respectively. Intracellular lactate levels were then measured using an intracellular lactate assay kit. Figure 2As shown in Figure B, 2-DG inhibits lactate production. Lactate production depends on the balance between glycolysis and mitochondrial metabolism. We wanted to investigate whether the activities of enzymes involved in these two pathways could regulate lactate levels, and thus regulate the protein expression level of YY1. Oxamate can inhibit lactate production by regulating the activity of lactate dehydrogenase LDH. Conversely, rotenone promotes intracellular lactate production by inhibiting mitochondrial respiratory chain complex I, causing cells to shift to glycolytic metabolism. Figure 2 As shown in C. To investigate the effects of Oxamate and Rotenone on intracellular lactate production, we treated LCL cells with 10 mM Oxamate and Rotenone for 24 h and then measured intracellular lactate production. The results showed ( Figure 2 D) Oxamate inhibits lactate production, while Rotenone promotes intracellular lactate production. It has been confirmed that glucose promotes intracellular lactate production, while 2-DG inhibits it. Therefore, we treated BJAB, HeLa, and LCL cells with different concentrations of glucose or 2-DG for 24 hours, and Western blotting was used to detect the expression level of YY1 protein. Figure 2 As shown in Figure E, glucose treatment reduced YY1 protein expression levels in a dose-dependent manner, while 2-DG treatment had virtually no effect on YY1 protein expression levels. This indicates that glucose-induced lactate reduces YY1 protein expression levels in a dose-dependent manner. To investigate the effect of intracellular lactate regulated by Oxamate and Rotenone on YY1 protein levels, we treated BJAB, HeLa, and LCL cells with different concentrations of Oxamate and Rotenone for 24 h, respectively. Western blotting was used to detect YY1 protein expression levels. Figure 2 As shown in E, Rotenone-induced lactate reduces YY1 protein expression levels in a dose-dependent manner.

[0104] Example 3: Knocking out YY1 promotes the in vivo and in vitro tumorigenicity of HPV-positive and EBV-positive cells.

[0105] 1. The acidic lactate microenvironment promotes the in vitro colony formation ability of LCL cells by downregulating YY1 expression.

[0106] To investigate the impact of lactate-acid-mediated YY1 downregulation on tumor malignant proliferation, we selected EBV-treated B lymphocytes (LCL) for our study. We constructed two stable YY1 knockdown LCL cell lines and one stable knockdown control cell line with irrelevant sequences, namely LCL-shYY1-1, LCL-shYY1-2, and LCL-shCtrl. Western blot analysis confirmed the effects. Figure 3 As shown in Figure A, these three stable cell lines were successfully constructed, with LCL-shYY1-1 exhibiting better knockdown. The successful construction of stable YY1 knockdown LCL cell lines has been verified. Next, the effect of LA and NaLA-mediated YY1 downregulation on the in vitro colony-forming ability of LCL cells was investigated using a suspension cell soft agar colony formation assay. The initial cell numbers for LCL-shYY1-1, LCL-shYY1-2, and LCL-shCtrl were all 0.6 × 10³. Fresh medium was added every two days, along with 10 mM MLA and 10 mM NaLA, respectively. After two weeks of continuous culture, the results are shown in Figure A. Figure 3 As shown in Figure B, compared to the non-knockdown group (shCtrl), YY1 knockdown promoted the in vitro colony formation ability of LCL cells; compared to shYY1-2, shYY1-1 promoted the in vitro colony formation ability of LCL cells even more strongly; compared to the MOCK group, LA and NaLA treatments promoted the in vitro colony formation ability of LCL-shCtrl cells, significantly promoted the in vitro colony formation ability of LCL-shYY1-1 and LCL-shYY1-2 cells, and more significantly promoted the in vitro colony formation ability of LCL-shYY1-1 cells. This indicates that lactate-acidic environment-mediated downregulation of YY1 expression promotes the in vitro colony formation ability of LCL cells, and the lower the YY1 expression level, the stronger the colony formation ability of LCL cells.

[0107] 2. The acidic lactate microenvironment promotes the malignant proliferation of EBV-related B-cell lymphomas by downregulating YY1 expression.

[0108] Since LCL cells are derived from EBV-infected wild-type cells, we used LCL-Luci-shYY1 and LCL-Luci-shCtrl cell lines, with the same cell number (1x10⁷), to intraperitoneally inject NOD / SCID mice to construct an EBV-related B-lymphoma NOD / SCID mouse intraperitoneal tumor model. To simulate the formation of the tumor microenvironment in mice, NaLA (0.5 g / kg) was injected intraperitoneally every two days, with the same volume of PBS injected intraperitoneally as a control. The results showed ( Figure 3 C) Four weeks after intraperitoneal injection, LCL-Luci-shYY1 showed enhanced tumorigenicity and growth in the peritoneal cavity of NOD / SCID mice compared to LCL-Luci-shCtrl (FC: 25.1 vs 35.6); compared to the MOCK group, the NaLA group showed significantly enhanced tumorigenicity and growth in the peritoneal cavity compared to the PBS group (FC: 25.1 vs 61.2; 35.6 vs 165). This result is consistent with the results of in vitro clonogenic assays of LCL cells. Combined with in vivo and in vitro tumorigenicity assays of LCL cells, the results indicate that the lactic acidic microenvironment promotes the malignant proliferation of EBV-related B-cell lymphomas by downregulating YY1 expression.

[0109] 3. The lactic acid-acidic microenvironment promotes the in vitro clone-forming ability of HeLa cells by downregulating YY1 expression.

[0110] The above experiments show that the acidic environment of lactate promotes the in vitro colony formation, invasion, and migration of LCL cells by downregulating YY1 expression, and promotes the in vivo tumorigenesis and growth of EBV-related B lymphocytes. To investigate the effect of lactate-mediated YY1 downregulation on the malignant proliferation of solid tumors, we selected HPV-infected cervical cancer cells (HeLa) as the research object. We constructed two stable YY1 knockdown HeLa cell lines and one stable knockdown control cell line with irrelevant sequences, namely HeLa-shYY1-1, HeLa-shYY1-2, and HeLa-shCtrl. Western blot analysis confirmed the effects. Figure 4 As shown in Figure A, these three stable cell lines were successfully constructed, with Hela-shYY1-1 showing better knockdown. Further, the effect of LA and NaLA-mediated YY1 downregulation on the in vitro colony-forming ability of Hela cells was investigated using a plate colony formation assay. The initial cell numbers for Hela-shYY1-1, Hela-shYY1-2, and Hela-shCtrl were all 0.6 × 10³. Fresh culture medium was added every two days, along with 20 mM MLA and 20 mM NaLA, respectively. After two weeks of continuous culture, the results are shown in Figure A. Figure 4 As shown in Figure B, compared to shCtrl, knocking down YY1 promoted the proliferation of HeLa cells; compared to HeLa-shYY1-2, HeLa-shYY1-1 showed slightly stronger colony-forming ability; compared to the MOCK group, LA and NaLA treatments promoted the in vitro colony-forming ability of HeLa-shYY1-1 cells. This indicates that lactate-acidic environment-mediated downregulation of YY1 expression promotes the in vitro colony-forming ability of HeLa cells.

[0111] 4. The acidic lactic acid microenvironment promotes the malignant proliferation of HPV-related cervical cancer by downregulating YY1 expression.

[0112] Since HeLa cells are derived from HPV-positive human cervical cancer tissue, we selected two cell lines, HeLa-Luci-shYY1-1 and HeLa-Luci-shCtrl, with the same cell number (1×10⁻⁶). 7 NaLA was mixed with Martrigel and subcutaneously injected into BALB / cNude mice to establish a subcutaneous tumor model of HPV-related cervical cancer in BALB / cNude mice. To simulate the formation of the tumor microenvironment in the mouse tumors, NaLA (1 g / kg) was injected intratumorally every two days, with the same volume of PBS injected intratumorally as a control. The results showed that... Figure 4C) Compared to Hela-Luci-shCtrl, Hela-Luci-shYY1 showed enhanced subcutaneous tumorigenesis and growth in BALB / cnude mice, and the enhanced tumorigenesis and growth in the NaLA group was significantly higher than that in the PBS group. This result is consistent with the results of in vitro Hela cell clonogenic experiments. Combined with the in vivo and in vitro tumorigenesis experiments of Hela cells, the results indicate that the lactic acidic microenvironment promotes the malignant proliferation of HPV-related cervical cancer by downregulating YY1 expression.

[0113] 1) Steps for plate colony formation experiment:

[0114] HPV-positive cervical cancer HeLa cells were selected and treated with lactate (LA) and sodium lactate (NaLA).

[0115] a. Seed 0.3 × 10³ HeLa cells into a six-well plate, add 2 mL of LDM medium, and incubate in an incubator.

[0116] b. Replace the culture medium with fresh medium every 2 days, adding 20mM MLA or 20mM NaLA each time the medium is replaced.

[0117] c. After 14 days of culture, when obvious clonal colony formation is observed, the culture is terminated and crystal violet staining is performed.

[0118] d. Discard the culture medium and wash gently once with 1×PBS. Add 2 mL of 4% paraformaldehyde and fix at room temperature in the dark for 15 min.

[0119] e. Gently wash once with 1×PBS, add 2 mL of 0.1% crystal violet, and stain at room temperature in the dark for 15 min.

[0120] f. Gently wash three times with 1×PBS, discard the 1×PBS, air dry, and then scan the image using a high-quality scanner.

[0121] 2) Suspension cell tumorigenesis-soft agar colony formation experiment steps:

[0122] a. Prepare a 4% low melting point agar solution: Weigh 0.8g of low melting point agar powder and dissolve it in 20ml of ultrapure water, then autoclave.

[0123] b. Place a 4% low melting point agar solution in a preheated 56°C water bath.

[0124] c. Preheat RPMI 1640 medium containing 10% FBS (containing 10 mM MLA or 10 mM NaLA).

[0125] d. Quickly prepare 0.75% lower agar using preheated culture medium. Take 2 mL of the well-prepared 0.75% lower agar and add it to a six-well plate, avoiding the formation of air bubbles. Let it stand at room temperature until it solidifies.

[0126] e. Prepare 2 mL of 0.36% agar using 10% FBS-RPMI 1640 medium containing 0.6 x 10³ suspension cells, and add it to the top layer of the solidified 0.75% agar.

[0127] f. Allow to stand at room temperature until solidified. After solidification, transfer to a 37°C cell culture incubator for further culture.

[0128] g. During the period, appropriate fresh 1640 medium (containing 10 mM MLA or 10 mM NaLA) may be added as needed to continue culturing.

[0129] h. Once obvious clonal colony formation is observed after about 2-3 weeks of culture, the culture can be terminated, and excess culture medium in the agar medium can be aspirated.

[0130] i. Prepare a 0.1% crystal violet staining solution containing 2% ethanol, and stain at room temperature in the dark for 2 hours.

[0131] Wash gently with 1×PBS several times until the clonal colonies are clearly visible.

[0132] 3) BALB / cNude subcutaneous tumor model

[0133] a. Purchase 4-5 week old female BALA / cNude mice, 5 mice per cage, and acclimatize them in a clean-grade mouse house for one week.

[0134] b. Based on weight, the mice were randomly divided into 4 groups, and each mouse was numbered and marked by piercing its ears.

[0135] c. Culture HeLa-shCtrl and HeLa-shYY1 cells at a rate of 1×10⁷ cells per tumor.

[0136] d. Wash cells twice with 1×PBS, resuspend 1×10⁷ cells per tumor-bearing cell in 200L of serum-free and antibiotic-free DMEM medium, mix with 200L of high-concentration Matrigel, transfer to sterile EP tubes, place on ice, and prepare for subcutaneous injection in mice.

[0137] e. Properly grasp the mouse and disinfect the injection site with an alcohol swab. Inject the prepared mixture of cells and Matrigel into the subcutaneous tissue on the mouse's back.

[0138] f. Continue feeding, injecting sodium lactate (1g / kg mouse body weight) into the tumor every two days, and injecting the same volume of 1×PBS as a control.

[0139] g. Weigh and record the mice weekly, and carefully observe their growth.

[0140] 4) NOD / SCID in vivo tumorigenesis model in mice

[0141] a. Purchase 4-5 week old female NOD / SCID mice, 5 mice per cage, and acclimatize them in a clean-grade mouse house for one week.

[0142] b. The mice were randomly divided into 4 groups according to their weight, and each mouse was labeled with a number using picric acid.

[0143] c. Culture LCL-shCtrl and LCL-shYY1 cells at a rate of 1×10⁷ cells per mouse.

[0144] d. Wash the cells twice with 1×PBS, resuspend 1×10⁷ cells per mouse in 200L of 1×PBS, transfer to sterile EP tubes, place on ice, and prepare for intraperitoneal injection in mice.

[0145] e. Properly grasp the mouse and disinfect the injection site with an alcohol swab. Inject the cells into the mouse through the abdomen using an intraperitoneal injection method.

[0146] f. Inject sodium lactate (1 g / kg mouse body weight) intraperitoneally every two days, and inject the same volume of 1×PBS as a control.

[0147] g. Tumor signals in mice were detected at week 0 and week 4 after cell injection using the IVIS spectrum Imaging System (PerkinElmer).

[0148] h. The substrate D-luciferin (150 mg / kg) was injected intraperitoneally. Imaging was performed 12 min after injection. Tumor size was detected using the IVIS spectrum imaging system (PerkinElmer), and fluorescence values ​​at an exposure time of 0.5 s were analyzed.

[0149] Example 4: Lactic acid (LA) promotes the translocation of YY1 from the nucleus to the cytoplasm.

[0150] To demonstrate that lactate (LA) promotes the translocation of YY1 cells from the nucleus to the cytoplasm, we performed an indirect immunofluorescence experiment, such as... Figure 5 A visual demonstration shows that lactic acid-induced regulation of YY1 expression occurs under acidic conditions. Treatment with 10 mL A for 24 hours reduces YY1 expression in the nuclei of KSHV-treated BCBL1 cells, KSHV, and EBV double-negative DG75 cells, while increasing YY1 expression in the cytoplasm. Using YY2 cells as a control, 10 mL A treatment for 24 hours does not affect the nucleoplasmic localization of YY2. Figure 5 Treatment with 10 mL A for 24 hours reduced YY1 expression in the nucleus of EBV-infected cells B95.8 and LCL, and KSHV and EBV double-negative cells BJAB, while increasing YY1 expression in the cytoplasm. Using YY2 as a control, 10 mL A treatment for 24 hours did not affect the nucleoplasmic localization of YY2. Figure 5 C showed that 10 mM MLA did not affect the nuclear-cytoplasmic localization of YY1 cells in HPV-infected HeLa cervical cancer cells, while treatment with 20 mM and 30 mM MLA for 24 h caused changes in the nuclear-cytoplasmic localization of YY1 cells, and the nuclear-cytoplasmic localization of YY2 cells was not affected by LA. Figure 5 D showed that 20 mL MLA treatment decreased YY1 expression in the HeLa cell nucleus and increased YY1 expression in the cytoplasm. This indicates that LA promotes the translocation of YY1 from the nucleus to the cytoplasm.

[0151] Immunofluorescence of suspension cells

[0152] Wash the prepared slides once with 75% ethanol, twice with 1×PBS, and incubate with 0.1% FSG for 15 min. Carefully aspirate the supernatant and air dry. Collect the suspended cells into 15 mL centrifuge tubes, centrifuge at 1200 rpm for 3 min, resuspend the cells in 1×PBS and wash 2-3 times. Carefully aspirate the supernatant.

[0153] Cells were fixed with 4% paraformaldehyde at room temperature in the dark for 25 min, centrifuged at 1200 rpm for 3 min, and the supernatant was carefully discarded. Cells were washed twice with 1×PBS, centrifuged at 1200 rpm for 3 min, and the supernatant was carefully discarded. Cells were resuspended in 10 μL of 1×PBS, dropped onto a prepared glass slide, gently spread, and incubated at 37°C for 40 min.

[0154] Add 1 mL of blocking buffer and block for about 20 minutes. Meanwhile, prepare the primary antibody. Dilute the primary antibody in the blocking buffer according to the ratio, centrifuge at 14000 rpm for 2 minutes to remove impurities, and spot 50 μl onto the sealing film. Place the coverslip with the cell side down to bind with the primary antibody on the sealing film, and incubate at room temperature for 1 hour.

[0155] Place the slide back into the six-well plate with the cell side facing up, and wash three times with blocking buffer.

[0156] Preparation of secondary antibody: Dilute the secondary antibody (1:500) in blocking buffer, centrifuge at 14000 rpm for 2 min to remove impurities, aspirate 50 μl of supernatant, spot it onto the sealing membrane, incubate the cells with the secondary antibody facing down at room temperature for 1 hr.

[0157] Place the slide back into the 6-well plate with the cell side up, wash twice with blowing buffer, and then wash once with 1×PBS.

[0158] Mix 1×PBS with DAPI (1:1000), centrifuge at 14000 rpm for 2 min, add 1 mL to each well, stain in the dark for 5 min, wash 2-3 times with 1×PBS, and air dry the slides in the dark in sequence.

[0159] Mounting: Add 20 μl of mounting medium to a glass slide, place the coverslip with the cell-containing side down on the mounting medium, and seal with nail polish. Observe the experimental results under a fluorescence microscope.

[0160] Immunofluorescence of adherent cells

[0161] First, aspirate the culture medium from the adherent cells, wash the cells 2-3 times with 1×PBS, and carefully discard the supernatant. Fix the cells with 4% paraformaldehyde at room temperature in the dark for 20 minutes, and carefully discard the supernatant. Wash the cells 2-3 times with 1×PBS, and carefully discard the supernatant.

[0162] Add 1 mL of blocking buffer and block for approximately 15 minutes. Wait for the primary antibody to be prepared. The remaining steps are the same as for immunofluorescence of suspension cells.

[0163] Nucleoplasmic separation

[0164] Collect adherent cells in 25 cm² Flask containers, centrifuge at 800 rpm for 3 min. Discard the supernatant, resuspend in pre-chilled 1×□PBS, centrifuge at 800 rpm for 3 min, wash twice, and discard the supernatant.

[0165] Add 200 μL of cytoplasmic lysis buffer containing four protease inhibitors, gently resuspend the cells, place on ice for 5 min, centrifuge at 12,000 rpm for 5 min at 4°C. Transfer the supernatant to another pre-chilled 1.5 mL EP tube; this contains cytoplasmic proteins.

[0166] Add 100 μL of nuclear lysis buffer to the untransferred cell pellet, resuspend the cells, place on ice for 5 min, centrifuge at 12000 rpm for 5 min at 4°C. Transfer the supernatant to a 1.5 mL EP tube; this is the nuclear lysis buffer.

[0167] Example 5: Prediction and Identification Analysis of YY1 Protein Acid Response Sequence

[0168] Based on the demonstration in Example 4 that YY1 responds to an acidic environment and translocates to the cytoplasm, analysis of its amino acid sequence and prediction of its nucleus exit sequence revealed two clusters of acidic or basic amino acid sequences (LAR1, mE and LAR2, mH, respectively) and three potential nucleus exit sequences (NES1, NES2, and NES3, respectively). Figure 6 As shown in Figure A. To further confirm the existence of the YY1 protein acid response sequence, we constructed different YY1 sequence mutants with RFP tags and transfected them into 293, MM, and HeLa cells, respectively. The results showed that the YY1 protein acid response sequence was mainly located in the LAR2 region ( Figure 6 B, 6C, 6E), and statistical analysis was performed on the responses in 293, MM, and HeLa cells. Figure 6 E). Further analysis of the stability of each YY1 mutant revealed that, under the influence of LA, YY1-mH, YY1-NES2, and YY1-NES3 showed higher stability than YY1-mE, YY1-wt, and YY1-NES1. Figure 6 F), and its stability was statistically analyzed (F). Figure 6 G). To further confirm that the LAR2 region of YY1 is indeed the acid-responsive region of YY1 in response to an acidic environment, three HeLa cell lines stably expressing YY1-wt, YY1-mH, and YFP were selected and cultured with different concentrations of LA. Immunofluorescence experiments showed that after the LAR2 mutation in the acid-responsive region of YY1 (YY1-mH), LA could not induce nucleation in YY1 cells. Figure 6 H), further confirming that the acid-responsive sequence of YY1 is LAR2. The polypeptide corresponding to this sequence is called YY1-MH.

[0169] Example 6: LAR2 mutation in the YY1 protein acid-responsive sequence inhibits the in vitro colony formation ability of HeLa cells:

[0170] To further illustrate how the acid-responsive sequence of YY1 mediates nuclear degradation under acidic conditions, thereby promoting tumor formation, we conducted a plate colony formation experiment. Hela-YFP, Hela-YY1-wt, and Hela-YY1-mH cells were cultured in LA and NaLA, respectively, with an initial seeding number of 0.3 × 10⁻⁶ cells. 3 and 0.6×10 3 The culture medium was replaced with fresh medium every two days, and 20 mM MLA and 20 mM NaLA were added respectively. The culture was continued for two weeks, and the results were as follows: Figure 7As shown, compared to HeLa-YFP, overexpression of YY1-wt or YY1-mH inhibited the colony-forming ability of HeLa cells; compared to the MOCK group, LA and NaLA treatments significantly enhanced the in vitro colony-forming ability of HeLa-YFP cells, slightly enhanced the in vitro colony-forming ability of HeLa-YY1-wt cells, and had no effect on the in vitro colony-forming ability of HeLa-YY1-mH. This indicates that the lactate-acidic environment-mediated downregulation of YY1 expression promotes the in vitro colony-forming ability of HeLa cells through the acid-responsive sequence LAR2 of YY1.

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[0258] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims. sequence list <110> Fudan University <120> A novel target for the diagnosis and treatment of infectious and non-infectious malignant tumors and its interventional applications <160> 10 <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <400> 1 Glu Glu Glu Glu Glu Asp Asp Asp Asp Glu Asp 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <400> 2 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <400> 3 His His His His His His His His His His His 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <400> 4 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 1 5 10 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Ile Val Glu Leu His Glu Ile Glu Val 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <400> 6 Ala Val Glu Ala His Glu Ala Glu Ala 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <400> 7 Ile Lys Thr Leu Glu Gly Glu Phe Ser Val 1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <400> 8 Ala Lys Thr Ala Glu Gly Glu Ala Ser Ala 1 5 10 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <400> 9 Leu Asp Phe Asn Leu Arg Thr His Val Arg Ile 1 5 10 <210> 10 <211> 11 <212> PRT <213> Artificial Sequence <400> 10 Ala Asp Ala Asn Leu Arg Thr His Ala Arg Ala 1 5 10

Claims

1. The application of a reagent for recognizing YY1 protein in the preparation of in vitro diagnostic reagents, characterized in that, The aforementioned in vitro diagnostic reagents are used to predict tumor grade or prognosis. The amino acid sequence of the YY1 protein at positions 70-80 is shown in SEQ ID NO 3; The tumor in question is either EBV-induced lymphoma or HPV-induced cervical cancer. The reagent used to identify the YY1 protein is an antibody or its antigen-binding fragment.

2. The use of a YY1 protein or a mutant thereof in the preparation of a medicament for treating tumors, characterized in that, The amino acid sequence of the YY1 protein at positions 70-80 is shown in SEQ ID NO 3; The amino acid sequence of the mutant YY1 protein at positions 70-80 is shown in SEQ ID NO 4; The tumor in question is either EBV-induced lymphoma or cervical cancer caused by HPV.

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